Processes and intermediates for the preparations of carboprost and carboprost tromethamine, and carboprost tromethamine prepared therefrom

ABSTRACT

The invention relates to processes for preparing Carboprost or Carboprost Tromethamine, and intermediates prepared from the process, and 5,6-trans isomer free Carboprost or Carboprost Tromethamine prepared therefrom. The invention also relates to a novel crystalline form of Carboprost Tromethamine.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a DIVISIONAL of U.S. patent application Ser. No.17/408,864 filed on Aug. 23, 2021 the disclosure of which is herebyincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel processes and intermediates forthe preparations of Carboprost and Carboprost Tromethamine, and a novelhigh melting point crystal of Carboprost Tromethamine preparedtherefrom.

BACKGROUND OF THE INVENTION

Carboprost and Carboprost Tromethamine (INN, trade names Hemabate,Tham), as shown in the following Scheme A, are both syntheticprostaglandin analogues of PGF_(2α) (specifically, it is15-methyl-PGF_(2α)) with oxytocic properties. Carboprost and CarboprostTromethamine can induce contractions and trigger abortion in earlypregnancy, and can also reduce postpartum bleeding.

Although the current regulations restrict the content of impurities inactive pharmaceutical ingredients (APIs) more and more stringently,almost all of the commercially available Carboprost and CarboprostTromethamine still contain about 3% 5,6-trans isomers and about 2%15(R)-epimers. The following Scheme B illustrates the chemicalstructures of Carboprost and isomers thereof, i.e., 5,6-trans Carboprostand 15-epi Carboprost. It appears that the methods for mass productionof Carboprost or Carboprost Tromethamine in industry have some problemsto be solved, especially in constructing the steric orientations of thecis-double bond at C5-C6 position and the tertiary alcohol at C15position of Carboprost or Carboprost Tromethamine. It is also obviousthat the current purification methods for removing the impurities ofCarboprost or Carboprost Tromethamine are not effective and have to beimproved.

C15-(R/S)-selectivity

Carboprost Tromethamine is the original product of Upjohn. The firstscalable synthesis of Carboprost Tromethamine was described by chemistsof Upjohn (Yankee et al., J. Am. Chem. Soc, 96(18), 5865-5876, 1974). Asshown in the following Scheme C (1), the 15-methyl substituent wasconstructed from the benzoyl γ-lactone-enone of Formula a withtrimethylaluminum or with methylmagnesium bromide. However, theselectivity of the 15(S)-product was only 50% in both cases, which meansthat the process has no selectivity. WO 2008/081191 discloses that thealkylation of triethylsily γ-lactone-enone of Formula b andmethylmagnesium chloride can obtain 15(S)-product with a highestselectivity of 70%, as shown in the following Scheme C (2). WO2017/093770 discloses that the alkylation of p-phenylbenzoylγ-lactone-enone of Formula c and methylmagnesium bromide can obtain15(5)- product with a selectivity of only 55%, and it also discloses theuse of various chiral additives in order to increase the selectivity ofthe 15(S)-product, and found that the addition of (S)- Taddol canincrease the selectivity to 70%, as shown in the following Scheme C (3).However, the highest selectivity disclosed in WO 2017/093770 is at mostthe same as that disclosed in WO 2008/081191.

C5,6-(trans/cis)-selectivity

WO 2008/081191 discloses that carrying out the Wittig reaction atambient temperature and in the solvent of dimethyl sulfoxide (DMSO) asdisclosed by Yankee et al., will generate 6 to 8% undesired 5,6trans-isomer, as shown in the following Scheme D (1). WO 2008/0181191further discloses that the reaction at a lower temperature, i.e., from−5° C. to +5° C., can reduce the content of the 5,6 trans-isomer toabout 3%, as shown in the following Scheme D (2). Given the above, sincethe commercially available Carboprost and Carboprost Tromethamine allcontain about 3% 5,6-trans isomer, it seems that changing the reactionconditions of the Wittig reaction may be useless in further reducing thecontent of 5,6-trans isomer generated therefrom.

Removing 15(R)-Epimer and Trans Isomer by Purification of CarboprostMethyl Ester

As disclosed by Yankee et al. and in Eur. J. Pharm, Sci, 3, 27-38(1995), the allylic tertiary alcohol at C15 position of Carboprost isvery unstable; thus, just a few acids or a little heat will result inproducing a large amount of 15(R)-epimer rapidly via epimerization. Dueto this reason, the current methods of mass production of Carboprost orCarboprost Tromethamine in industry do not construct the stereochemistryof allylic tertiary alcohol in the intermediates formed in early steps,since the stereochemistry of the allylic tertiary alcohol is difficultto be maintained until the final stage. Therefore, almost all of thecurrent methods of mass production of Carboprost or CarboprostTromethamine in industry construct the stereochemistry of the allylictertiary alcohol until forming the structure of the final product or inthe intermediates formed in late steps, or remove the undesired15(R)-epimer and trans isomer after forming the final product.

Although the final product Carboprost Tromethamine is a crystallinesolid at room temperature, it is still very difficult to efficientlyremove the 15(R)-epimer and trans isomer simultaneously only bycrystallization purification. In addition, since the polarity ofCarboprost is very large, it is more difficult to remove the15(R)-epimer and trans isomer from Carboprost by chromatography.

Consequently, the isomers generated during the formation of Carboprostor Carboprost Tromethamine cannot be effectively removed, and there isno any suitable late-step intermediate used for separating and removingisomers in the prior art methods. In order to purify the final products,two additional steps are required to create a late-step intermediatethat is suitable for use in separation and removal of isomers. Asdisclosed in the prior art references, e.g., J. Am. Chem. Soc., 96(18),5865-5876, 1974; WO 2008/081191; WO 2017/093770; CN 111777537; and CN102816099, Carboprost has to be esterified to form Carboprost methylester, then the 15(R)-epimer and trans isomer of Carboprost methyl esterare removed by chromatography, and the Carboprost methyl ester ishydrolyzed into Carboprost with higher purity, but the yield and purityin such method are very unsatisfactory. CN 1136938 C discloses the useof expensive and special simulated moving bed (SMB) chromatography toseparate and remove the isomers of Carboprost methyl ester, but thepurity of the final product is still not good enough. CN 102816099discloses the use of analytical-grade HPLC column chromatography packing(5 μm) for separation, but the trans isomers still cannot be removedcompletely. In addition, since the amount that can be separated by themethod of CN 102816099 is small, such method is difficult to be used forindustrial-grade mass production.

SUMMARY OF THE INVENTION

Given the above, there is a need to discover and develop a method forpreparing high purity Carboprost or Carboprost Tromethamine, which cansignificantly reduce the formation of impurities or isomers including15(R)-epimer and trans isomer, and can also effectively remove thegenerated impurities or isomers.

The objective of the present invention is to provide an efficientprocess for producing Carboprost or Carboprost Tromethamine. The processincludes a macrolactonization step, which can effectively remove5,6-trans isomer. The process also includes a methylation step ofmecrolactone-enone for constructing the orientation of tertiary alcoholat C15 position of Carboprost. The 15(S)-selectivity of methylation ofmacrolactone-enone is much higher than that of methylation ofγ-lactone-enone in all previous examples. The present invention alsoprovides a novel purification process that can effectively remove allisomers and can produce Carboprost and Carboprost Tromethamine moreefficiently, thereby forming final products with high purity, highmelting point, and good stability.

In one aspect, the present invention provides a process for preparinghigh purity Carboprost or Carboprost Tromethamine containing no morethan about 1% total isomers from a compound of Formula 1a containingabout 1 to 10% 5,6-trans isomer:

wherein

or a protecting group for carbonyl groups; and P₁ and P₂ are H orprotecting groups for hydroxy groups.

In another aspect, the present invention provides a process forpreparing high purity Carboprost or Carboprost Tromethamine containingno more than about 1% total isomers from a compound of Formula 1bcontaining about 1 to 10% 5,6-trans isomer,

wherein P₁ and P₂ are H or protecting groups for hydroxy groups.

In another aspect, the present invention provides a process forpurification of Carboprost, which is effective to purify low purityCarboprost containing a large amount of isomers, such as about 1 to 10%5,6-trans isomer and about 1 to 50% 15(R)-epimer, to a high purityCarboprost containing no more than about 1% total isomers.

In one another aspect, the present invention provides amacrolactone-enone intermediate of Formula 2a′, which is a novelintermediate for preparing Carboprost or Carboprost Tromethamine,

wherein P₁ is H or a protecting group for hydroxy groups.

In another aspect, the present invention provides a process forpreparing a macrolactone-tertiary alcohol of Formula 3, which is anintermediate for preparing Carboprost or Carboprost Tromethamine,

wherein P₁ is H or a protecting group for hydroxy groups, from amacrolactone-enone of Formula 2a′ with a methylation reagent.

In one another aspect, the present invention provides a high meltingpoint (106.4±1° C.) crystal of Carboprost Tromethamine having an X-raypowder diffraction (XRPD) pattern exhibiting characteristic peaks at thefollowing 2θ reflection angles: 6.9±0.2°, 10.3±0.2°, 18.8±0.2°, and21.9±0.2°.

In yet another aspect, the present invention provides a process forpreparing a high melting point crystal of Carboprost Tromethamine,comprising the use of a solvent of anhydrous acetonitrile.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the X-ray powder diffraction (XRPD) pattern of theCarboprost Tromethamine crystal of the present invention.

FIG. 2 shows the differential scanning calorimetry (DSC) thermogrampattern of the Carboprost Tromethamine crystal of the present invention.

FIG. 3 shows the X-ray powder diffraction (XRPD) patterns of theCarboprost Tromethamine crystals for five different batches according tothe present invention.

FIG. 4 shows the differential scanning calorimetry (DSC) thermogrampatterns of the Carboprost Tromethamine crystals for five differentbatches according to the present invention.

FIG. 5 shows the differential scanning calorimetry (DSC) thermogrampatterns of the Carboprost Tromethamine crystals prepared from Example 9of present invention.

FIG. 6 shows the differential scanning calorimetry (DSC) thermogrampatterns of the Carboprost Tromethamine crystals prepared from Example10 of present invention.

DETAILED DESCRIPTION OF THE INVENTION Definition

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butis also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or unless the alternatives are mutually exclusive,although the disclosure supports a definition that refers only toalternatives and “and/or.” Throughout this application, the term “about”is used to indicate that a value includes the inherent variation oferror for the device, the method being employed to determine the valueor the variation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

In the depiction of the compounds given throughout this description, awedged bold bond (

) means a bond projecting above the plane of the paper; a wedged hashedbond (

) means a bond projecting below the plane of the paper; and a wavy bond(

) means a bond projecting almost half above and half below the plane ofthe paper.

When used herein, the term “high purity Carboprost or CarboprostTromethamine,” “high purity Carboprost” or “high purity CarboprostTromethamine” means that Carboprost and/or Carboprost Tromethamine inquestion contains no more than about 1% total isomers, preferably nomore than about 0.8% total isomers or no more than about 0.5% totalisomers, and more preferably no more than about 0.3% total isomers. Theisomers or impurities indicated herein include 5,6-trans isomers,15(R)-epimers, and any other stereoisomers.

When used herein, the term regarding substantially free of 5,6-transisomer or the like means that a compound in question does not containmore than about 0.5%, about 0.3%, about 0.2%, about 0.1%, about 0.05%,or about 0.03% of impurities or isomers, such as 5,6-trans isomer, orcontains a none-detectable level of impurities or isomers, such as5,6-trans isomer measured by HPLC, for which the detection limit is notmore than about 0.03%.

Unless otherwise specified, the term “protecting group for hydroxygroups” has the meaning conventionally defined in organic syntheticchemistry, i.e., a group capable of protecting a hydroxyl group ormoiety of a compound against the attacks of a chemical reaction.Examples of the hydroxyl protecting group include, but are not limitedto, methoxymethyl, methoxythiomethyl, 2-methoxyethoxymethyl,bis(2-chloroethoxy)methyl, tetrahydropyranyl, tetrahydrothiopyranyl,4-methoxytetrahydropyranyl, 4-methoxytetrahydrothiopyranyl,tetrahydrofuranyl, tetrahydrothiofuranyl, 1-ethoxyethyl,1-methyl-1-methoxyethyl, triphenylmethyl, allyl, benzyl, substitutedbenzyl, acetyl, substituted acetyl, benzoyl, substituted benzyl, andSiR_(a)R_(b)R_(c) wherein R_(a), R_(b) and R_(c) are each independentlyunsubstituted or substituted alkyl or unsubstituted or substituted aryl,such as C₁₋₄ alkyl, phenyl, benzyl, substituted phenyl, and substitutedbenzyl.

Unless otherwise specified, the term “protecting group for carbonylgroups” has the meaning conventionally defined in organic syntheticchemistry, i.e., a group capable of protecting a carbonyl group ormoiety of a compound against the attacks of a chemical reaction.Examples of the carbonyl protecting group include, but are not limitedto, dialkyl ketal, diaralkyl ketal, diacetyl ketal, dithio ketal,1,3-dioxane, 1,3-dioxolane, 1,3-dithiane, 1,3-dithiolane, and1,3-oxathiolane. Preferred protecting groups for carbonyl groups includedialkyl ketal, 1,3-dioxane, and 1,3-dioxolane.

Each of the above mentioned groups such as alkyl and aryl may optionallybe substituted with one or more substituents selected from the groupconsisting of halogen, alkyl, aryl, alkoxyl, aryloxy, thioalkoxyl,thioaryloxy, alkylamino, arylamino, cyano, alkoxycarbonyl, arylcarbonyl,arylaminocarbonyl, alkylaminocarbonyl, and carbonyl, or a heterocyclicgroup selected from the group consisting of pyridinyl, thiophenyl,furanyl, imidazolyl, morpholinyl, oxazolinyl, piperidinyl, piperazinyl,tetrahydropyranyl, pyrrolidinyl, pyrrolidinonyl, and the like. Unlessotherwise specified, the term “alkyl” used herein refers to a straightor branched hydrocarbon group containing 1 to 8, 1 to 6, or 1 to 4carbon atoms, such as methyl, ethyl, isopropyl, tert-butyl, and thelike; or a cyclic saturated hydrocarbon group having 3 to 10 or 3 to 8carbon atoms, such as cyclopropyl, cyclopentyl, cyclohexyl, and thelike. The term “aryl” used herein refers to a monocyclic or polycyclicaromatic hydrocarbon radical, and having 6 to 20, 6 to 18, or 6 to 12carbon atoms, such as phenyl, naphthyl, anthryl, phenanthryl and thelike.

Synthesis of High Purity Carboprost from Known ProstaglandinIntermediates of Formula 1a or Formula 1b

According to the present invention, the high purity Carboprost orCarboprost Tromethamine can be prepared according to the reactions shownin Scheme 1 and Scheme 2:

The compound of Formula 1a in Scheme 1, wherein

or a protecting group for carbonyl groups; and P₁ and P₂ are protectinggroups for hydroxy groups, is a well-known prostaglandin F_(2α)intermediate. The compound of Formula 1b in Scheme 2, wherein P₁ and P₂are protecting groups for hydroxy groups, is also a well-known 15-methylprostaglandin F_(2α) intermediates. The two prostaglandin intermediatescan be prepared from a famous intermediate, Corey lactone, via a Wittigreaction. Due to different reaction conditions of the Wittig reaction,the two prostaglandin intermediates almost all contain about 1% to about10% 5,6-trans isomer.

As shown in Step (1) of Scheme 1 and Step (1) of Scheme 2, themacrolactonization reaction may involve the activation of the carboxylor/and hydroxyl functional groups. In this route, the macrolactonizationreaction comprises the initial formation of a thioester with a suitablereagent, which includes, but is not limited to, S-pyridin-2-ylchloromethanethioate, 2,2′-dipyridyl disulfide/triphenylphosphine, or4-tert-butyl-2-(2-(4-tert-butyl-1-isopropyl-1H-imidazol-2-yl)disulfanyl)-1-isopropyl-1H-imidazole/triphenylphosphine.

The macrolactonization reaction may alternatively involve the initialformation of a mixed anhydride with a suitable reagent in the presenceor absence of a base or a Lewis acid. Suitable reagents for forming themixed anhydrides include, but are not limited to, 2,4,6-trichlorobenzoylchloride, 2-nitro-6-nitrobenzoic anhydride, p-nitrofluoromethylbenzoicanhydride, p-nitrobenzoic anhydride, and the like. Examples of thesuitable bases include 4-(dimethylamino)pyridine, pyrolidinopyridine,triethylamine, N,N-diisopropylethylamine, and isopropyldiethylamine.Examples of suitable Lewis acids include Sc(OTf)₃, TiCl₄, AgClO₄,trimethylsilyl chloride (TMSCl) and TiCl₂(OTf).

The macrolactonization reaction can also be achieved by using acondensation reagent and a base in an appropriate solvent. Suitablecondensation reagents include, but are not limited to,N,N′-dicyclohexylcarbodiimide, 2-chloro-1-methyl-pyridium iodide,2-chloro-4,5-dihydro-1,3-dimethyl-1H-imidazolium chloride,N,N-diphenylchlorophenylmethyleniminium chloride, cyanuric chloride,1,3-dimethyl-2-chloroimidazolium chloride,N,N,N,N-tetramethylchloroformamidinium chloride, and the like. Examplesof suitable bases include pyridine, triethylamine,diisopropylethylamine, 4-dimethylaminopyridine (DMAP), and the like.Suitable solvents for the condensation reaction include methylenechloride, tetrahydrofuran, and 1,2-dichloroethane, and a mixturethereof.

Upon analyzing a resultant compound of Formula 2a, 2a′, 2b, 3 or 4 byHPLC or UPLC, it is unexpectedly found that the resultant compound ofFormula 2a, 2a′, 2b, 3 or 4, contains no more than about 0.1% of the5,6-trans isomer or less, which reveals that the macrolactonizationreaction exhibits a high cis-selectivity; that is, the 5,6-cis compoundsof Formula 1a or 1b dominate in the macrolactonization reaction whereasthe 5,6-trans compounds of Formula 1a or 1b hardly undergo themacrolactonization reaction.

Step (2) of Scheme 1 involves a deprotection and an oxidation of thecompound of Formula 2a, wherein

by removing the P₂ at the ω-side chain and/or the P₁. The conditions forcarrying out the deprotection reactions are obvious to persons skilledin the art. For example, the macrolactone of Formula 2a wherein P₁ andP₂ are tetrahydropyranyl protecting groups is dissolved in a suitablesolvent, such as methanol or a solvent mixture of acetone and water in avolumetric ratio of 5 to 1; treated with a deprotecting agent such ashydrogen chloride, p-toluenesulfonic acid, or pyridiump-toluenesulfonate; and stirred at room temperature for 10 minutes to 10hours. The reaction is quenched with a base, e.g., ammonium hydroxide orthe like, and subjected to a work-up procedure conducted in aconventional manner. The deprotected product of Formula 2a, wherein P₁and P₂ are H, is oxidated with a suitable oxidant such as MnO₂ or2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), to form themacrolactone-enone of Formula 2a′, wherein P₁ is H.

For example, the macrolactone of Formula 2a wherein P₁ is atetrahydropyranyl protecting group and P₂ is a tent-butyldimethylsilylprotecting group is dissolved in a suitable solvent, such astetrahydrofuran (THF); treated with a deprotecting agent such astetrabutylammonium fluoride (TBAF); and stirred at room temperature for10 minutes to 10 hours. The reaction is subjected to a work-up procedureconducted in a conventional manner. The deprotected product of Formula2a, wherein P₁ is a tetrahydropyranyl protecting group and P₂ is H, isthen oxidated with a suitable oxidant such as Collins oxidant, Swernoxidant, PCC oxidant, PDC oxidant, and TEMPO oxidant, preferably TEMPOoxidation, to form the macrolactone-enone of Formula 2a′, wherein P₁ isa tetrahydropyranyl protecting group.

Step (2) of Scheme 1 also involves a deprotection of the compound ofFormula 2a, wherein

is a protecting group for carbonyl groups. The conditions for carryingout the deprotection reactions are obvious to persons skilled in theart. For example, the macrolactone of Formula 2a wherein

is a 1,3-dioxane protecting group and P₁ is H, is dissolved in asuitable solvent, such as THF or acetone; treated with a deprotectingagent such as 1M HCl solution; and stirred at room temperature for 10minutes to 10 hours. The reaction is quenched with a base, e.g.,saturated NaHCO₃ solution or the like, and subjected to a work-upprocedure conducted in a conventional manner, to form themacrolactone-enone of Formula 2a′, wherein P₁ is H.

Moreover, Step (2) of Scheme 2 involves a deprotection of the compoundof Formula 2b, wherein P₂ is a protecting group for hydroxy groups, byremoving the P₂ at the ω-side chain and/or the P₁. The conditions forcarrying out the deprotection reactions are obvious to persons skilledin the art.

Step (3) of Scheme 1 shows a methylation of the macrolactone-enone ofFormula 2a′ wherein P₁ is a protecting group for hydroxy groups, to forma macrolactone-tertiary alcohol of Formula 3a. According to the presentinvention, the methylation agent includes, but is not limited to, MeLi,MeMgCl, MeMgBr, MeMgI, Me₃Al, or a mixture thereof. Preferably, themethylation agent is MeMgCl, MeMgBr, MeMgI, MeLi, or a mixture thereof.Most preferably, the methylation agent is MeLi. The non-limiting,suitable solvent used in the reaction can be selected fromtetrahydrofuran, ether, toluene, hexane, or a mixture thereof. Thereaction is carried out at a temperature ranging from about −120° C. tothe room temperature, preferably from about −100° C. to about −40° C.The methylation agent is used in an amount such that the reactants arecompletely reacted as monitored by thin layer chromatography (TLC).

It is unexpectedly found that the reaction of the macrolactone-enone anda methyl Grignard reagent as the methylation agent exhibits up to about65% 15(S)-selectivity. However, as shown in Scheme A (1), the15(S)-selectivity of the reaction of γ-lactone-enone and a methylGrignard reagent is only 50%. It appears that the structure ofmacrolactone is beneficial to the 15(S)-selectivity than γ-lactone. Inaddition, the present invention surprisingly shows that the use of a lowcost and more convenient MeLi as the methylation agent, the methylationof the macrolactone-enone can have a 15(S)-selectivity of about 75% ormore, which is very high and cannot be achieved even using a chiraladditive in the reaction of γ-lactone (WO 2017/093770, using (S)-Taddol,the highest selectivity is merely 70%).

Step (4) of Scheme 1 and Step (3) of Scheme 2 involve a purification ofthe macrolactone-tertiary alcohol of Formula 3, for removing15(R)-epimer. Typically, the separation of isomers of Carboprost is themost expensive and time-consuming step in the mass production ofcarboprost. However, the cost for separation of isomers of the presentinvention is much lower than the prior art techniques, e.g., J. Am.Chem. Soc., 96(18), 5865-5876, 1974; WO 2008/081191; WO 2017/093770; CN111777537; and CN 102816099A. The reasons are listed as follows:

-   (a) In the prior art techniques, Carboprost has to be esterified to    form an intermediate, Carboprost methyl ester, which is suitable for    use in removing isomers, and then the isomers of Carboprost methyl    ester can be removed by chromatographic purification. However, it is    unexpectedly found that late-step intermediate of Formula 3 is an    intermediate that is suitable for use in removing isomer; thus, an    additional esterification reaction is unnecessary to be used in the    present invention.-   (b) When using chromatographic purification to remove 5,6-trans    isomer and 15(R)-epimer of Carboprost intermediates, 5,6-trans    isomer (HPLC, RRT 0.93) is usually more difficult to be separated    than 15(R)-epimer (HPLC, RRT 0.88). CN 102816099 even uses an    analytical-grade HPLC with filler (5 μm) in order to remove    5,6-trans isomer (RRT 0.93) of Carboprost methyl ester. In contrast,    the intermediate of Formula 3, as well as the compound of Formula    2a′, formed after macrolactonization have substantially free of    5,6-trans isomer, thus, a general silica gel column chromatography    for industrial mass production can be simply used to separate    15(R)-epimer that is easier to be removed.-   (c) The methylation of macrolactone-enone of the present invention    exhibits higher selectivity, so the amount of the generated    15(R)-epimer is fewer and can be removed more easily.

In Step (4) of Scheme 1 and Step (3) of Scheme 2, the chromatographicpurification can be performed using ester-type, ether-type, ketone-typeor halogenated solvents, or the like or a mixture thereof. For using amixture of dichloromethane and acetone, good separation can also beachieved with ethyl acetate, isopropyl acetate, methyl tert-butyl ether,acetone, methyl ethyl ketone, or a mixture thereof. By chromatography,the amount of the undesired 15(R)-epimer can be decreased to a specifiedlimit, i.e., ≤about 0.5%, ≤about 0.3%, ≤about 0.2%, ≤about 0.1%, orless.

As shown in Step (5) of Scheme 1 and Step (4) of Scheme 2, themacrolactone-tertiary alcohol of Formula 3 is hydrolyzed to Carboprostin methanol solution by treatment with lithium hydroxide solution.Acidification to obtain Carboprost has to be performed quickly, to avoidepimerization in the acid medium.

Accordingly, the present invention provides a process for preparingCarboprost containing no more than 1% total isomers, the processcomprising the steps of:

-   -   (1) macrolactonization of a compound of Formula la containing 1        to 10% 5,6-trans isomer

wherein

or a protecting group for carbonyl groups; and P₁ and P₂ are H orprotecting groups for hydroxy groups, to form a compound of Formula 2a:

wheren

P₁ and P₂ are as defined above for Formula 1a;

-   -   (2) removing the protecting group for carbonyl groups of the        compound of Formula 2a, wherein

is a protecting group for carbonyl groups; or removing P₂ and/oroxidizing the compound of Formula 2a, wherein

to form a compound of Formula 2a′:

wherein P₁ is H or a protecting group for hydroxy groups;

-   -   (3) methylation of the compound of Formula 2a′ with a        methylation reagent, to form a compound of Formula 3:

wherein P₁ is H or a protecting group for hydroxy groups;

-   -   (4) chromatographic separation of the compound of Formula 3 to        remove isomers;    -   (5) hydrolysis of the compound of Formula 3, to form a compound        of Formula 4:

-   -   -   wherein P₁ is H or a protecting group for hydroxy groups;            and        -   optionally performing a deprotecting reaction of the            compound of Formula 2a, 2a′, 3, or 4, when P₁ is a            protecting group for hydroxy groups, to form a compound of            Formula 2a, 2a′, 3, or 4, wherein P₁ is H.

The present invention also provides a process for preparing high purityCarboprost containing no more than 1% total isomers, the processcomprising the steps of:

-   -   (1) macrolactonization of a compound of Formula 1b containing 1        to 10% 5,6-trans isomer and 1 to 50% 15(R)-epimer,

wherein P₁ and P₂ are H or protecting groups for hydroxy groups, to forma compound of Formula 3a:

wherein P₁ and P₂ are as defined above;

-   -   (2) performing a deprotection reaction of the compound of        Formula 3a, when P₁ is H or a protecting group for hydroxy        groups, and P₂ is a protecting group for hydroxy groups, to form        a compound of Formula 3a, wherein P₁ is H or a protecting group        for hydroxy groups, and P₂ is H;    -   (3) chromatographic separation of the compound of Formula 3a,        wherein P₁ is H or a protecting group for hydroxy groups, and P₂        is H, to remove isomers;    -   (4) hydrolysis of the compound of Formula 3a, to form a compound        of Formula 4:

wherein P₁ is H or a protecting group for hydroxy groups; and

-   -   (5) optionally performing a deprotecting reaction of the        compound of Formula 3a or 4, when P₁ is a protecting group for        hydroxy groups, to form a compound of Formula 3a or 4, wherein        P₁ is H.

Purification of a Low Purity Carboprost Which Contains an Excess Amountof Isomers

Crude Carboprost obtained from conventional reactions or due to anoverreaction of epimerization normally contains a large amount or anexcess amount of isomers. The inventors found that Carboprost containingan excess amount of isomers can be further purified by using the processof the present invention. The process of the present invention comprisesproviding a low purity Carboprost containing at least about 1 to 10%5,6-trans isomer and about 1 to 50% 15(R)-epimer; macrolactonization ofthe low purity Carboprost to form a macrolactone-tertiary alcohol;chromatographic separation of excess isomers of themacrolactone-tertiary alcohol; and hydrolysis of themacrolactone-tertiary alcohol, to form a high purity Carboprostcontaining no more than about 1% total isomers.

Accordingly, the present invention provides a process for purificationof Carboprost, comprising the steps of:

-   -   (1) macrolactonization of a low purity Carboprost containing 1        to 10% 5,6-trans isomer and 1 to 50% 15(R)-epimer, to form a        compound of Formula 3b:

-   -   (2) chromatographic separation of isomers of the compound of        Formula 3b; and    -   (3) hydrolysis of the compound of Formula 3b, to form a high        purity Carboprost containing no more than 1% total isomers.        Carboprost Salt Formation with Tromethamine

To form Carboprost Tromethamine from Carboprost, the process furthercomprises a salt formation with tromethamine, and crystallization ofCarboprost Tromethamine. The salt formation and crystallization stepsmay follow the method disclosed in CN 102336693 or WO 2017/093770. Ingeneral, Carboprost can be first dissolved in a suitable solvent, suchas acetone, acetonitrile, methanol, ethanol, isopropyl alcohol, and acombination thereof; then tromethamine in a suitable solvent, such aswater, methanol, ethanol, isopropanol, and a combination thereof, can beadded to the solution; and the mixture can be heated to about 85° C. forabout 1 hours to form a homogeneous solution. Thereafter, the homogenoussolution can be allowed to cool down to about 60° C., and a white solidslowly starts to precipitate. The reaction mixture can be further cooleddown to room temperature, and then filtered to obtain the white crystalof Carboprost Tromethamine. The melting point of the white crystal thusobtained is typically about 95° C. to 105° C. as originally disclosed byPfizer (e.g., the prescribing information of Hemabate® provided byPfizer).

In case of Example 1 of CN 102336693, about 1.20 g CarboprostTromethamine was precipitated during lowering the temperature from themixture of 100 ml acetonitrile and 0.5 ml water, and the melting pointof the white crystal is measured as 103.97° C. (see FIG. 2 ). Thepresent inventors repeated Example 1 of CN 102336693 and found that themelting point of the obtained Carboprost Tromethamine crystal ismeasured as 103.41° C.

In case of Example 1g of WO 2017/093770, about 593 g CarboprostTromethamine was precipitated from the mixture of isopropanol andacetone. WO 2017/093770 does not disclose the melting point of theCarboprost Tromethamine crystal obtained from Example 1g. The presentinventors repeated Example 1g of WO 2017/093770 and found that themelting point of the obtained Carboprost Tromethamine crystal ismeasured as 97.49° C.

The melting points of the above mentioned known Carboprost Tromethaminecrystals range from about 97° C. to about 104° C., which are indeedwithin the scope as originally provided by Pfizer (95° C. to 105° C.).

Recrystallization of Carboprost Tromethamine

The recrystallization on Carboprost Tromethamine may provide effects ondecreasing the content of 15(R)-isomer. The recrystallization methods ofCarboprost Tromethamine have been disclosed in CN 102336693 and WO2017/093770. The present inventors repeated the method CN 102336693disclosed using water with acetone, and found that the recrystallizedCarboprost Tromethamine has a melting point of 103.85° C. as measured.The present inventors also repeated the method WO 2017/093770 disclosedusing isopropanol with acetone, and found that the recrystallizedCarboprost Tromethamine has a melting point of 99.46° C. as measured.

It is an unexpectedly found that a specially high melting pointCarboprost Tromethamine crystal can be obtained by a recrystallizationprocess of Carboprost Tromethamine using a specific solvent of anhydrousacetonitrile. The high melting point Carboprost Tromethamine crystalthus formed has a differential scanning calorimetry (DSC) thermogrampattern comprising an endothermic peak with a peak maximum of 106.4±1.0°C., which is obviously higher than the upper limit of the melting pointrange originally provided by Pfizer and that of the above mentionedCarboprost Tromethamine crystals disclosed in the prior art.

Preparation of the High Melting Point Carboprost Tromethamine Crystal

The present invention provides a process for preparing a high meltingpoint crystalline form of Carboprost Tromethamine, comprising the stepsof:

-   a. adding Carboprost Tromethamine into anhydrous acetonitrile to    form a mixture, wherein the anhydrous acetonitrile is in an amount    of from about 80 to 250 ml per 1 g of Carboprost Tromethamine;-   b. heating the mixture to a temperature ranging from about 70° C. to    90° C. to give a homogeneous solution;-   c. cooling down the homogeneous solution to form the crystalline    form of Carboprost Tromethamine; and-   d. optionally isolating the crystallized product.

In some embodiments, the mixture can be heated to about 80° C. forcompletely dissolving Carboprost Tromethamine; then the homogenoussolution can be slowly cooled down, and a white solid is slowly startedto precipitate; and the mixture can be further cooled down to roomtemperature, and then filtered and dried to obtain the crystalline formof Carboprost Tromethamine.

In some embodiments, the amount of the anhydrous acetonitrile used instep a ranges from about 80 to 250 ml, about 100 to 220 ml, or about 150to 200 ml, per 1 g of Carboprost Tromethamine; the anhydrousacetonitrile has a water content of less than about 0.05%, less thanabout 0.01%, less than about 0.005%, or less than about 0.001% (w/w);and the process is preferably carried out without adding water. Thecrystalline form of Carboprost Tromethamine prepared from the processhas a differential scanning calorimetry (DSC) thermogram patterncomprising an endothermic peak with a peak maximum of 106.4±1.0° C.,which is higher than the other known crystalline forms of CarboprostTromethamine. Since the Carboprost Tromethamine crystal obtained by thepresent invention has the highest melting point compared to all otherknown Carboprost Tromethamine crystals, it is the most stablecrystalline form of Carboprost Tromethamine.

Moreover, the high melting point Carboprost Tromethamine crystal of thepresent invention has an X-ray powder diffraction (XRPD) patternexhibiting characteristic peaks at the following 2θ reflection angles:6.9±0.2°, 10.3±0.2°, 18.8±0.2°, and 21.9±0.2°, which is obviouslydifferent from the 2θ reflection angles: 6.6±0.2°, 9.9±0.2°, 18.5±0.2°,and 21.6±0.2° of the crystal disclosed in CN 102336693. It indicatesthat the high melting point Carboprost Tromethamine crystal is a novelcrystalline form of Carboprost Tromethamine.

In one embodiment of the present invention, the Carboprost Tromethaminecrystal has an XRPD pattern exhibiting characteristic peaks at thefollowing 2θ reflection angles: 6.9±0.2°, 10.3±0.2°, 18.8±0.2°, and21.9±0.2°. In a preferred embodiment, the XRPD pattern further comprisescharacteristic peaks at the following 2θ reflection angles: 9.1±0.2°,9.5±0.2°, 11.0±0.2°, and 20.5±0.2°. More preferably, the XRPD pattern ofthe Carboprost Tromethamine crystal is consistent with FIG. 1 . Theparticular data of the Carboprost Tromethamine crystal is shown in Table1.

TABLE 1 2θ d relative angle (°) value (Å) intensity (%) 6.92 12.76100.00 9.08 9.73 5.20 9.49 9.31 4.77 9.83 8.99 3.23 10.26 8.61 25.3711.02 8.02 2.76 12.52 7.06 1.34 13.16 6.72 2.55 13.61 6.50 2.37 13.976.33 1.59 14.48 6.11 2.23 16.10 5.50 3.91 16.84 5.26 4.47 16.97 5.225.06 17.41 5.09 1.93 17.72 5.00 4.65 17.99 4.93 7.48 18.43 4.81 8.9218.81 4.71 31.86 19.58 4.53 20.95 19.77 4.49 28.20 20.17 4.40 24.2520.35 4.36 17.83 20.54 4.32 12.33 21.07 4.21 8.03 21.42 4.15 6.27 21.864.06 16.88 22.77 3.90 2.06 23.57 3.77 2.88 23.98 3.71 2.97 24.25 3.673.18

In one embodiment, the present invention provides a crystalline form ofCarboprost Tromethamine having an XRPD pattern substantially as shown inFIG. 1 .

In one embodiment, the present invention provides the XRPD patterns ofthe Carboprost Tromethamine crystals for five different batches (a) to(e), as shown in FIG. 3 . The particular data of the four majorseparated characteristic peaks are clearly marked and shown in Table 2.The average positions of characteristic peaks are located at about 6.9°,10.3°, 18.8°, and 21.9°, respectively.

TABLE 2 Sample 2θ angle (°) (a) 6.87 10.21 18.80 21.88 (b) 6.92 10.2218.80 21.87 (c) 7.02 10.32 18.82 21.87 (d) 7.00 10.29 18.81 21.88 (e)6.91 10.25 18.82 21.90 Average 6.94 10.26 18.81 21.88

The XRPD characteristic peak positions of the Carboprost Tromethaminecrystal of the present invention locate at higher 2-theta (2θ) angle(6.9±0.2°, 10.3±0.2°, 18.8±0.2°, and) 21.9±0.2°, comparing with6.6±0.2°, 9.9±0.2°, 18.5±0.2°, and 21.6±0.2° disclosed by CN102336693.The characteristic peak at 6.621° of Carboprost Tromethamine disclosedby CN 102336693 represents a d-spacing of 13.3397 Å. On the other hand,the characteristic peak at 6.92° of Carboprost Tromethamine of thepresent invention represents a d-spacing of 12.76 Å. The more than 0.5 Åd-spacing difference is a very clear evidence indicating that themolecule packing are different in crystal lattice structure. This resultshows that the Carboprost Tromethamine crystal is a novel crystallineform with a more stable and denser ordering packing structure comparingto the previous arts.

In one embodiment, the present invention provides a CarboprostTromethamine crystal having a DSC thermogram pattern comprising anendothermic peak with a peak onset temperature of approximately 103.9°C. and a peak maximum of approximately 106.4±1.0° C. In a preferredembodiment, the present invention provides a crystalline form ofCarboprost Tromethamine having a DSC thermogram pattern substantially asshown in FIG. 2 .

In one embodiment, the present invention provides the DSC thermogrampatterns of the Carboprost Tromethamine crystals for five differentbatches (a) to (e), as shown in FIG. 4 . The particular data of the peakmaximum for single endothermic peak is clearly marked and shown in Table3. The average peak maximum of these endothermic peaks is about 106.4°C.

TABLE 3 Peak Sample Maximum (° C.) (a) 106.27 (b) 106.63 (c) 106.44 (d)106.34 (e) 106.40 Average 106.42

The DSC peak maximum temperature of about 106.4° C. for the CarboprostTromethamine crystal of the present invention is far beyond thedisclosed melting point of Carboprost Tromethamine of 95 to 105° C. (asoriginally provided by Pfizer) and the peak maximum temperature of103.97° C. disclosed in CN 102336693, indicating that the CarboprostTromethamine crystal of the present invention is a novel crystal withhigher thermal stability comparing to the previous arts. It iswell-known for person skilled in the art that a crystal comprising thehighest melting point is the most stable crystalline form inthermodynamics; hence, the novel Carboprost Tromethamine crystal of thepresent invention is the most stable one compared to the previous arts.

The United States Pharmacopeia recommends that Carboprost Tromethamineshould be stored in a freezer (−20° C.). In contrast, the presentinvention proves that the high melting point crystalline form ofCarboprost Tromethamine is very stable even being treated at 20° C. for6 months, 5° C. for 18 months, and 80° C. for 3 days. Hence, thestability of the Carboprost Tromethamine crystal of present inventionhas been significantly improved.

All of the compounds and/or processes disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compounds and process of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutions andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

EXAMPLES

X-ray Powder Diffraction (XRPD) Analysis: The XRPD patterns werecollected on a Bruker D2 PHASER diffractometer with fixed divergenceslits and 1D LYNXEYE detector. The samples (ca. 100 mg) were flatlyplaced on a sample holder. The prepared samples were analyzed over a 2θrange from 5° to 40° with step size of 0.02 degrees and step time of 1second using CuK_(α) radiation at a power of 10 mA and 30 kV. TheCuK_(β) radiation was removed by a divergent beam nickel filter.

Differential Scanning calorimetry (DSC) Analysis: The DSC thermogrampatterns were collected on a TA DISCOVERY DSC25 instrument. The sampleswere weighed into an aluminum pan with a crimping closed aluminum lid.The prepared samples were analyzed from 25° C. to 150° C. at scan rateof 10° C./min under a flow of nitrogen (ca. 50 ml/min). The meltingtemperature and heat of fusion were calibrated by indium (In) beforemeasurement. The melting point of all samples was determined by the peakmaximum of the endothermic peak during DSC measurement in thisinvention.

Example 1(8aR,9R,10R,11aS,Z)-10-((tert-butyldimethylsilyl)oxy)-9-((S,E)-3-((tert-butyldimethyl-silyl)oxy)oct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one

The 1.4 kg of7-((1R,2R,3R,5S)-3-((tert-butyldimethylsilyl)oxy)-2-((S,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)-5-hydroxycyclopentyl)hept-5(Z)-enoicacid, which includes about 6.5% of 5,6-trans isomer determined bydetecting it's deprotected product in HPLC analysis, and 475 g ofpyridine were dissolved in 6.5 L of dichloromethane at ambienttemperature under nitrogen. Then, 843 g of benzoyl chloride was addedinto the mixture and stirred for one hour. The reaction mixture waschecked by TLC to confirm completion of the reaction. The mixture wasquenched with 6 L of saturated sodium bicarbonate aqueous solution andstirred for 10 minutes. The solution was stand to separate into twophases, and an organic layer was collected. The organic layer wasfurther evaporated off. The concentrated residue was diluted with 6 L ofToluene, and washed with 0.1N hydrochloric acid aqueous solution andbrine, respectively. The organic layer was collected and evaporated offto obtain crude silyl-protected 1,9-lactone compound. The crude compoundwas further purified by chromatography on silica gel using a mixture ofhexane and ethyl acetate as a gradient eluent. Yield of the obtainedsilyl-protected 1,9-lactone was 1.0 kg (74%).

The 0.1 g of product was further processed with hydrolysis reaction anddeprotection reaction to obtain the crude product. HPLC analysis showedthat no 5,6-trans isomer was detectable for the crude compound.

¹H-NMR (CDCl₃): δ 5.156˜5.595 (m, 5H), 4.067 (q, 1H), 3.809 (q, 1H),1.247˜2.532 (m, 20H), 0.852˜0.898 (m, 21H), −0.005˜0.034 (m, 12H);¹³C-NMR (CDCl₃) : δ 173.620, 136.520, 131.169, 129.135, 127.890, 72.809,72.088, 55.345, 44.567, 41.599, 38.669, 36.134, 31.831, 26.730, 26.571,25.895, 25.835, 25.349, 25.136, 22.624, 18.222, 18.063, 14.047, −4.252,−4.556, −4.579, −4.768; MS (m/z, EI): Calculated for C₃₂H₆₀O₄Si2Na (M⁺):587.4 and 587.5 found.

Example 2(8aR,9R,10R,11aS,Z)-9-((S,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)-10-((tetra-hydro-2H-pyran-2-yl)oxy)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one

The 2.0 g of7-((1R,2R,3R,5S)-2-((S,E)-3-((tert-butyldimethylsilyl)oxy)oct-1-en-1-yl)-5-hydroxy-3-((tetrahydro-2H-pyran-2-yl)oxy)cyclopentyl)hept-5(Z)-enoicacid, which includes 1.8% of 5,6-trans isomer, determined by detectingit's deprotected compound in HPLC analysis, and 0.72 g of pyridine weredissolved in 20 ml of dichloromethane at ambient temperature undernitrogen. Then, 1.27 g of benzoyl chloride was added into the mixtureand stirred for 30 minutes. The reaction mixture was checked by TLC toconfirm completion of the reaction. The mixture was quenched with 15 mlof saturated sodium bicarbonate aqueous solution and stirred for 10minutes. The solution was stand to separate into two phases, and anorganic layer was collected. The organic layer was evaporated off, andthe crude ether-protected 1,9-lactone compound was obtained. The crudecompound was purified by chromatography on silica gel using a mixture ofhexane and ethyl acetate as a gradient eluent. Yield of the obtainedether-protected 1,9-lactone was 1.35 g (70%).

The 0.1 g of product was further processed with hydrolysis reaction anddeprotection reaction to obtain the crude product. HPLC analysis showedthat no 5,6-trans isomer was detectable for the crude compound.

¹H-NMR (CDCl₃): δ 5.172˜5.630 (m, 5H), 4.813˜4.944 (m, 1H), 3.428˜4.091(m, 4H), 1.230˜2.619 (m, 26H), 0.845˜0.879 (m, 12H), 0.012˜0.038 (m,6H); ¹³C-NMR (CDCl₃) : δ 173.635 (173.559), 136.778 (136.619), 131.260,129.666, (129.507), 127.640, 95.390 (94.623), 81.933 (77.910), 73.159,62.457 (61.106), 53.971 (53.106), 44.962 (44.855), 39.618, 38.662(38.578), 37.417, 31.816 (31.778), 30.639 (30.601), 26.730, 25.417,25.319, 24.962, 22.601, 19.732 (19.573), 18.237 (18.222), 14.009,−4.252, −4.267, −4.806, −4.844; MS (m/z, EI): Calculated forC₃₁H₅₄O₅SiNa (M⁺): 557.4 and 557.4 found.

Example 3(8aR,9R,10R,11aS,Z)-9-((E)-3-oxooct-1-en-1-yl)-10-((tetrahydro-2H-pyran-2-yl)oxy)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one

The 1.5 g of7-((1R,2R,3R,5S)-5-hydroxy-2-((E)-3-oxooct-1-en-1-yl)-3-((tetrahydro-2H-pyran-2-yl)oxy)cyclopentyl)hept-5(Z)-enoicacid and 0.68 g of pyridine were dissolved in 15 ml of dichloromethaneat ambient temperature under nitrogen. Then, 1.21 g of benzoyl chloridewas added into the mixture and stirred for 30 minutes. The reactionmixture was checked by TLC to confirm completion of the reaction. Themixture was quenched with 10 ml of saturated sodium bicarbonate aqueoussolution and stirred for 10 minutes. The solution was stand to separateinto two phases, and an organic layer was collected. The organic layerwas evaporated off, and the crude 1,9-lactone compound was obtained. Thecrude compound was purified by chromatography on silica gel using amixture of hexane and ethyl acetate as a gradient eluent. Yield of theobtained 1,9-lactone was 1.02 g (71%).

¹H-NMR (CDCl₃): δ 6.688 (ddd, 1H), 6.235 (dd, 1H), 5.326 (dt, 1H), 5.197(br s, 2H), 4.592 (t, 0.5H), 4.550 (t, 0.5H), 4.060 (dd, 0.5H), 3.952(dd, 0.5H), 3.692˜3.800 (m, 1H), 3.388˜3.447 (m, 1H), 1.236˜2.684 (m,26H), 0.887 (t, 3H); ¹³C-NMR (CDCl₃): δ 200.466 (200.299), 173.499(173.408), 146.569 (146.417), 131.951, 131.784, 126.873, 96.321(96.612), 81.386 (78.790), 72.445 (72.111), 62.646 (61.736), 54.130(53.546), 45.030 (44.939), 40.582 (40.309), 39.808 (38.024), 35.998,31.451 (31.421), 30.677 (30.601), 26.715, 25.364 (25.303), 23.952,22.442, 19.550, 18.988, 13.895; MS (m/z, EI): Calculated for C₂₅H₃₈O₅Na(M+Na⁺): 441.3 and 441.3 found.

Example 4(8aR,9R,10R,11aS,Z)-10-hydroxy-9-((S,E)-3-hydroxyoct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one

The 480 g of(8aR,9R,10R,11aS,Z)-10-((tert-butyldimethylsilyl)oxy)-9-((S,E)-3-((tert-butyldimethyl-silyl)oxy)oct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one(from Example 1) was dissolved in 4.5 L of tetrahydrofuran at ambienttemperature. Then, 775 g of tetrabutylammonium fluoride trihydrate wasadded into the solution. The homogenous solution was heated at 45° C.and stirred for 6 hours. The reaction mixture was checked by TLC toconfirm completion of the reaction. The mixture was cooled down atambient temperature and subsequently quenched with 8 L of saturatedsodium bicarbonate aqueous solution. The solution was stand to separateinto two phases, and an organic layer was collected. The organic layerwas evaporated off to obtain crude 1,9-lactone diol compound. The crudecompound was purified by chromatography on silica gel using a mixture ofhexane and ethyl acetate as a gradient eluent. Then, the diol compoundwas further crystallized in mixed solvents of ethyl acetate and hexane.Yield of the obtained crystal of diol compound was 259 g (91%).

¹H-NMR (CDCl₃): δ 5.203˜5.642 (m, 5H), 4.409 (q, 1H), 3.800 (q, 1H),3.304 (br s, 1H), 1.281˜2.608 (m, 21H), 0.868 (t, 3H); ¹³C-NMR (CDCl₃) :δ 173.437, 136.785, 131.973, 131.411, 127.464, 76.065, 73.158, 71.936,56.201, 45.074, 40.285, 37.196, 36.050, 31.655, 26.722, 26.532, 25.272,25.158, 22.562, 13.971; MS (m/z, EI): Calculated for C₂₀H₃₀O₄ (M⁺):336.2 and 336.2 found.

Example 5(8aR,9R,10R,11aS,Z)-10-hydroxy-9-((E)-3-oxooct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one

The 300 g of(8aR,9R,10R,11aS,Z)-10-hydroxy-9-((S,E)-3-hydroxyoct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclo-penta[b]oxecin-2(3H)-one(from Example 4) was dissolved in 3 L of tetrahydrofuran at ambienttemperature, and 2,3-dichloro-5,6-di-cyano-1,4-benzo-quinone was added.The reaction mixture was heated at 55° C. and stirred for 2 hours. Aftercompletion of reaction, the mixture was cooled down at ambienttemperature and evaporated off Then, the dark-brown residue was dilutedwith dichloromethane, and yellow solid was precipitated. The mixture wasfiltered off, and the filtrate was further concentrated to obtain acrude ketone compound. The crude compound was purified by chromatographyon silica gel using a mixture of hexane and ethyl acetate as a gradienteluent. Yield of the obtained 15-keto 1,9-lactone was 276 g (93%).

¹H-NMR (CDCl₃): δ 5.183˜6.686 (m, 5H), 3.991˜4.062 (m, 1H), 1.251˜2.642(m, 21H), 0.887 (t, 3H);¹³C-NMR (CDCl₃) : δ 200.101, 173.316, 145.536,132.064, 131.737, 126.865, 76.383, 72.406, 56.239, 45.591, 40.983,40.839, 36.027, 31.435, 26.729, 25.302, 23.754, 22.433, 13.895; MS (m/z,EI): Calculated for C₂₀H₃₀O₄ (M⁺): 334.2144 and 334.2130 found.

Example 6 (8aR,9R,10R,11aS,Z)-10-hydroxy-9-((S,E)-3-hydroxy-3-methyloct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one

Method A: The 275 g of(8aR,9R,10R,11aS,Z)-10-hydroxy-9-((E)-3-oxooct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one(from Example 5) was dissolved in 4.5 L of tetrahydrofuran at ambienttemperature under nitrogen and cooled down at −70° C. The 1.0 L ofMethyllithium (2M in ether) was slowly added to the reaction mixture at−70° C., and the reaction was checked by TLC. After completion ofreaction, the mixture was quenched with saturated ammonium chlorideaqueous solution and stirred for 10 minutes. Then, 1 L of ethyl acetatewas added to the reaction mixture and allowed to warm up at ambienttemperature. The mixture was stand to separate into two phases, and anorganic layer was collected and dried over anhydrous Sodium sulfate.Subsequently, the solid was filtered off and the filtrate was evaporatedoff. The crude 15-methyl mixture compound was obtained and tested byHPLC. The ratio of 15-R/15-S of the crude compound is about 25/75. Thecrude compound was further purified by chromatography on silica gelusing a mixture of dichloromethane and acetone as a gradient eluent.Yield of the obtained pure 15-methyl compound was 163 g (57%).

Method B: The 2 g of(8aR,9R,10R,11aS,Z)-10-hydroxy-9-((E)-3-oxooct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one(from Example 5) was dissolved in 20 ml of tetrahydrofuran at ambienttemperature under nitrogen and cooled down at −70° C. Then, 21 ml ofmethylmagnesium bromide (1M in THF) was slowly added to the reactionmixture at −70° C. and warmed up at 0° C. The mixture was checked by TLCto confirm completion of reaction. Sampling the reaction mixture tocheck the ratio of 15-R/15-S of the product is about 35/65.

¹H-NMR (CDCl₃): δ 5.203˜5.713 (m, 5H), 3.787 (q, 1H), 1.167˜2.535 (m,25H), 0.859 (t, 3H); ¹³C-NMR (CDCl₃): δ 173.399, 140.709, 131.336,127.556, 127.526, 76.331, 72.840, 72.187, 56.233, 45.281, 42.875,40.317, 36.029, 32.173, 27.520, 26.693, 26.579, 25.274, 23.801, 22.541,13. 950; MS (m/z, EI): Calculated for C₂₁H₃₂O₃ (M⁺-H₂O): 332.2351 and332.2349 found.

Example 7(8aR,9R,10R,11aS,Z)-10-((tert-butyldimethylsilyl)oxy)-9-((S,E)-3-hydroxy-3-methyloct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one

The 2.0 g of(8aR,9R,10R,11aS,Z)-10-((tert-butyldimethylsilyl)oxy)-9-((E)-3-oxooct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-onein 20 ml of tetrahydrofuran at ambient temperature and cooled down at−70° C. The 3.5 ml of methyllithium (2M in ether) was slowly added tothe reaction mixture at −70° C., and the reaction was checked by TLC.After completion of reaction, the mixture was quenched with saturatedammonium chloride aqueous solution and stirred for 10 minutes. Then, 1ml of ethyl acetate was added to the reaction mixture and allowed towarmed up at ambient temperature. The mixture was phase separated, andan organic layer was dried over anhydrous sodium sulfate. Subsequently,the solid was filtered off, and the filtrate was evaporated off toobtain a crude 15-methyl mixture compound. The crude compound waspurified by chromatography on silica gel using a mixture ofdichloromethane and acetone as a gradient eluent. Yield of the obtainedpure 15-methyl compound was 1.05 g (51%).

¹H-NMR (CDCl₃): δ 5.168˜5.689 (m, 5H), 3.807 (q, 1H), 1.258˜2.551 (m,24H), 0.857˜0.944 (m, 12H), 0.011 (s, 6H);¹³C-NMR (CDCl₃): δ 173.582,140.368, 131.275, 127.746, 127.678, 76.954, 72.938, 72.005, 55.610,44.483, 42.844, 41.569, 36.127, 32.279, 28.081, 26.738, 26.487, 25.789,23.839, 22.579, 18.070, 14.040, −3.592, −4.518, −4.624.

MS (m/z, EI): Calculated for C₂₇H₄₈O₄SiNa (M+Na⁺): 487.3 and 487.3found.

Example 8(Z)-7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((S,E)-3-hydroxy-3-methyloct-1-en-1-yl)cyclo-pentyl)hept-5-enoicAcid, Carboprost

The 143 g of(8aR,9R,10R,11aS,Z)-10-hydroxy-9-((S,E)-3-hydroxy-3-methyloct-1-en-1-yl)-4,5,8,8a,9,10,11,11a-octahydrocyclopenta[b]oxecin-2(3H)-one(from Example 6) was dissolved in 750 ml of methanol, and 1.5 L of 1Nlithium hydroxide aqueous solution was added. The reaction mixture washeated at 60° C. for 2 hours. After completion of reaction, the mixturewas cooled down at room temperature, and pH value of the solution wasadjusted to about 8.5. The mixture was concentrated to remove methanoland the residue was further purified by acid-base extraction. Yield ofthe obtained Carboprost was 129 g.

¹H-NMR (CDCl₃): δ 5.278˜5.610 (m, 7H), 4.112˜4.133 (m, 1H), 3.866˜3.911(m, 1H), 1.252˜2.298 (m, 23H), 0.843 (t, 3H);

¹³C-NMR (CDCl₃): δ 177.316, 138.903, 129.431, 129.226, 128.839, 77.599,73.333, 72.339, 55.292, 50.427, 42.731, 42.488, 33.031, 32.257, 26.966,26.245, 25.099, 24.507, 23.809, 22.610, 14.063;

MS (m/z, EI): Calculated for C₂₁H₃₄O₄(M⁺-H₂O): 350.2 and 350.3 found

Example 9 Carboprost Tromethamine Salt Formation2-amino-2-(hydroxymethyl)propane-1,3-diol;(Z)-7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((S,E)-3-hydroxy-3-methyloct-1-en-1-yl)cyclopentyl)hept-5-enoate,Carboprost Tromethamine

Method A:

The 114 g of Carboprost was dissolved in 1.2 L of acetonitrile at 60°C., and the solution was heated to 65° C. The 37.4 g of tromethamine in120 ml water was slowly added into the solution. The reaction mixturewas further heated to 85° C. for reflux for 10 minutes. The homogenoussolution was allowed to cool down to 60° C., and the white solid wasslowly started to precipitate. The mixture was further cooled down toroom temperature and stirred over 16 hours. The reaction mixture wasfiltered to obtain white crystalline form of Carboprost Tromethamine(118 g). The melting point of the crystal was measured by DSC as 103.41°C., as shown in FIG. 5(a).

¹H-NMR (D₂O): δ 5.336˜5.594 (m, 4H), 4.109 (br s, 1H), 3.819 (br s, 1H),3.632 (s, 6H), 1.193˜2.427 (m, 23H), 0.764 (t, 3H);

¹³C-NMR (CDCl₃): δ 183.433, 138.523, 130.584, 129.203, 128.854, 75.928,73.576, 71.109, 71.109, 61.257, 59.451, 54.327, 41.971, 41.902, 37.136,31.565, 26.601, 25.850, 25.743, 24.779, 23.277, 21.888, 13.288:

MS (m/z, ESI): Calculated for C₂₅H₄₈NO₈ (MH⁺): 490.3374 and 490.3384found.

Method B:

The Carboprost Tromethamine crystal was prepared from an acetone/aqueoussolution by following the method disclosed in Example 3 of CN 102336693.The melting point of the crystal was measured by the inventor as 103.22°C., as shown in FIG. 5(b).

Method C:

The Carboprost Tromethamine crystal was prepared from an ether/aqueoussolution by following the method disclosed in Example 7 of CN 102336693.The melting point of the crystal was measured by the inventor as 103.43°C., as shown in FIG. 5(c).

Method D:

The Carboprost Tromethamine crystal was prepared from anisopropanol/acetone solution by following the method disclosed inExample 1g of WO 2017/093770. The melting point of the crystal wasmeasured by the inventor as 97.49° C., as shown in FIG. 5(d).

Example 10 Recrystallization of Carboprost Tromethamine

Method A:

10.0 g Carboprost Tromethamine was dissolved in 1 L acetonitrilecontaining water of 0.02% at 85° C. to give a homogeneous solution andthen cooled down the homogeneous solution to 60° C., and the white solidwas slowly started to precipitate. The mixture was further cooled downto room temperature. The resulting precipitate crystal was filtered anddried. After recrystallization, yield of the obtained high purityCarboprost Tromethamine crystal was 9.0 g. The melting point of thecrystal was measured by DSC as 106.40° C., as shown in FIG. 6(a). UponHPLC analysis, 15(R)-epimer was in an amount of 0.05%, and 5,6-transisomer was not detected.

Method B:

The Carboprost Tromethamine crystal was prepared from an acetone/aqueoussolution by following the method disclosed in Example 2, 4, 6, or 8 ofCN 102336693. The melting point of the crystal was measured by theinventor as 103.85° C., as shown in FIG. 6(b).

Method C:

The Carboprost Tromethamine crystal was prepared from anisopropanol/acetone solution by following the method disclosed inExample 1h of WO2017093770. The melting point of the crystal wasmeasured by the inventor as 99.46° C., as shown in FIG. 6(c).

Example 11 Purification of Low Purity Carboprost Tromethamine

3 g of commercially available Carboprost Tromethamine, which includes2.5% of 5,6-trans isomer and 1.5% 15(R)-epimer, was dissolved in acidicwater with pH of 3 and extracted with ethyl acetate to obtain 2 g ofCarboprost. A solution of Carboprost in 8 ml of anhydrous, oxygen-freexylene was treated with 1.73 g of 2,2′-dipyridyl disulfide and 2.06 g oftriphenylphosphine. After stirring for 18 hours at 25° C., the mixturewas diluted with 500 ml of xylene and heated at reflux for 4 hours. Thereaction mixture was further evaporated to remove the solvent, and theresidue was partitioned between a cold sodium bicarbonate aqueoussolution and ethyl acetate. The organic layer was collected and washedwith brine, and dried with anhydrous sodium sulfate. The mixture wasfiltered and concentrated to obtain crude 1,9-lactone compound. Thecrude compound was purified by chromatography on silica gel using amixture of dichloromethane and acetone as a gradient eluent. Yield ofthe obtained 15-methyl 1,9-lactone was 1.67 g.

The 1,9-lactone product was further processed by a hydrolysis reactionto obtain Carboprost which includes 2.5% of 15(R)-epimer but nodetectable 5,6-trans isomer in HPLC analysis. Carboprost Tromethaminewas formed from the Carboprost and tromethamine according to the processof Example 9, and crystallized and recrystallized according to theprocess of Example 10 (Method A) and Example 11 to obtain high purityCarboprost Tromethamine. Upon HPLC analysis, 15(R)-epimer was in anamount of 0.15%, and 5,6-trans isomer was not detected.

Example 12 Stability of Carboprost Tromethamine Crystal

The stability data shown in Tables 4 and 5 shows that the high meltingpoint Carboprost Tromethamine crystal is stable even being treated at20° C. for 6 months, 5° C. for 18 months, and 80° C. for 3 days. Thehigh melting point Carboprost Tromethamine crystal is very stable atroom temperature, even at higher temperature, so that the formation ofdegradation impurities can be effectively avoided.

TABLE 4 Carboprost Carboprost Carboprost Tromethamine TromethamineTromethamine Specimen crystal crystal crystal Conditions Control 25°C./60% RH 25° C./60% RH 1 month 6 months Purity 99.75% 99.71% 99.75%15(R)-epimer  0.25%  0.27%  0.25% 5,6-trans isomer ND ND ND TotalImpurities  0.25%  0.29%  0.25% Conditions 5° C. 5° C. 5° C.   1 month  12 months   18 months Purity 99.72% 99.75% 99.75% 15-epi  0.28%  0.25% 0.25% 5,6-trans isomer ND ND ND Total Impurities  0.28%  0.25%  0.25%

TABLE 5 Carboprost Carboprost Carboprost Tromethamine TromethamineTromethamine Specimen crystal crystal crystal Conditions Control 80° C.80° C.  1 days  3 days Purity 99.79% 99.79% 99.78% 15-epi  0.21%  0.20% 0.21% 5,6-trans isomer ND ND ND Total Impurities  0.21%  0.21%  0.22%

On the other hand, the hygroscopicity of the high melting pointCarboprost Tromethamine crystal prepared from Example 10 (Method A) andthe low melting point Carboprost Tromethamine crystal prepared fromExample 10 (Method B) was measured. The samples were placed in glassvial with 99% RH at 25° C. for 3 hours, and the water content of thesamples were measured by Karl Fischer titration, as shown in Table 6.The high melting point Carboprost Tromethamine crystal shows arelatively low rate of water absorption compared to the low meltingpoint one, indicating that the high melting point CarboprostTromethamine crystal is more stable and can be stored for a longer timeunder high humidity condition, so the high melting point CarboprostTromethamine crystal is more conducive to product handling, storage andshipping.

TABLE 6 Example 10 Example 10 (Method A) (Method B) high melting lowmelting Specimen point point Conditions Water Content (%) Original0.36571% 0.33029% 25° C./99% RH/1 h 1.50916% 2.23611% 25° C./99% RH/2 h3.35425% 5.44951% 25° C./99% RH/3 h 5.88095% 7.51338%

1-11. (canceled)
 12. A crystalline for of Carboprost Tromethamine,having a differential scanning calorimetry (DSC) thermogram patterncomprising an endothermic peak with a peak maximum of 106.4±1.0° C., andhaving an X-ray powder diffraction (XRPD) pattern comprisingcharacteristic peaks at the following 2θ reflection angles: 6.9±0.2°,10.3±0.2°, 18.8±0.2°, and 21.9±0.2°.
 13. The crystalline form ofCarboprost Tromethamine according to claim 12, wherein the XRPD patternfurther comprises characteristic peaks at the following 2θ reflectionangles: 9.1±0.2°, 9.5±0.2°, 11.0±0.2°, and 20.5±0.2°.
 14. Thecrystalline form of Carboprost Tromethamine according to claim 12,containing less than 0.1% 5,6-trans isomer.
 15. The crystalline form ofCarboprost Tromethamine according to claim 14, containing less than0.03% 5,6-trans isomer.
 16. A process for preparing the crystalline formof Carboprost Tromethamine according to claim 12, comprising the stepsof: (a) adding Carboprost Tromethamine into anhydrous acetonitrile toform a mixture, wherein the anhydrous acetonitrile is in an amount offrom 80 to 250 ml per 1 g of Carboprost Tromethamine; (b) heating themixture to a temperature ranging from 70° C. to 90° C. to give ahomogeneous solution; and (c) cooling down the homogeneous solution toform the crystalline form of Carboprost Tromethamine.
 17. The processaccording to claim 16, wherein the anhydrous actonitrile has a watercontent of less than 0.05%.